Effect of Helichrysum plicatum Subs. plicatum on Fructose-induced Nonalcoholıc Fatty Liver

Introduction
Nonalcoholic fatty liver disease (NAFLD) has been observed in alcohol-abstaining individuals. It is also associated with hypertriglyceridemia and insulin resistance. NAFLD comprises several stages of liver disease, from progression toward steatosis and steatohepatitis (NASH). They can eventually progress to liver cirrhosis. NAFLD is associated with hypertriglyceridemia, hypertension, dyslipidemia, insulin resistance, inflammation, lipid peroxidation, oxidative stress, and increased free-radical activity (Li et al., 2016) studies have highlighted the importance of calorie reduction and macronutrient composition (e.g, carbohydrate and fat) (Bernal et al., 2013).
With decreasing physical activity and increasing caloric intake from fast foods in industrialized countries, people’s dietary behaviors have changed, and the prevalence of metabolic diseases has increased. Fructose is an essential component of plant carbohydrates and a critical component of the human diet (Köseler et al., 2018; Abbasi et al., 2021). While average human fructose consumption was 16-20 g/d in the past, industrialization and new diet styles have increased fructose consumption to approximately 85-100 g/d (Drewnowski & Bellisle, 2007; Bergheim et al., 2008).
High fructose consumption causes insulin resistance and increases triglyceride levels (Bergheim et al., 2008). Fructose is taken up by fructokinase in the liver and is converted to fructose-1 phosphate, which is then converted to triose phosphate by aldolase. Thus, fructose passes three steps before glycolysis, according to glucosides. Interstingly, 60% conversion of fructose to fatty acids—more than that of glucose—increases lipid levels more than glucose. Increased levels of liver fats due to excess carbohydrate consumption have raised the prevalence of many liver-damaging diseases, including steatosis, steatohepatitis, advanced fibrosis, and sometimes cirrhosis (Bergheim et al., 2008; Ketaby & Mohammad-Sadegh, 2023). Many ethnobotanical remedies have been studied for their potential to alleviate NAFLD symptoms. However, none have demonstrated the desired results in rigorously controlled studies (Ebrahimi-Mameghani et al., 2014; Kołota & Głąbska, 2021).
Approximately 500 species of Helichrysum belonging to the Asteraceae family have been annotated worldwide. Twenty Helichrysum species have been identified in Turkey; 15 are endemic. They are named by different local names like “altın otu,’ “ölmez çiçek,’ “sarı çiçek,’ by different regions’ peoples in Turkey (Davis et al., 1988). Helichrysum plicatum subsp. plicatum (HPsP) is 4–42 cm tall and contains the highest levels of flavonoids among Helichrysum species. People in Turkey use it to treat wounds and burns, reduce kidney stones, enhance urinary function, and ameliorate ear pain (Ozturk et al., 2014; Aydin, 2020; Kültür et al., 2021).
Previous studies have demonstrated antimicrobial, antioxidant, anti-inflammatory, antidiabetic, and antimitotic properties of HPsP, probably due to its rich flavonoid content (Albayrak et al., 2010; Apaydin et al., 2017; Kramberger et al., 2021; Akinfenwa et al., 2021). The antilipidemic effects of HPsPs (grown in Anatolia) have not yet been studied on NAFLD. This study aimed to investigate the effects of HPsP on fatty acid composition and other biochemical parameters of NAFLD.

Materials and Methods

Animals and treatments
Theity-five 8-month-old male Wistar rats were obtained from the Fırat University Experimental Animal Research Center. The animals were housed in air-conditioned rooms on a 12:12 h dark: Light cycle with ad libitum access to rat chow and water. The animals were randomly divided into 5 groups, with 7 in each group. The control group (I) was fed a standard diet. Besides the standard diet, other groups were further treated as described. Group (II) received nutrients with extra HPsP (4 mg/kg by oral gavage). Group (III) was fed fructose (F) (40% w/v in drinking water). Group (IV) received nutrients with F and HPsP (4 mg/kg by gavage), and group (V) received nutrients with F and double dose HPsP (8 mg/kg by gavage). After 8 weeks of treatment, the rats were anesthetized with ether. Harvested tissues were stored at -85°C until further analysis. Vitamin levels were determined using high-performance liquid chromatography (HPLC), and fatty acid levels were determined using gas chromatography (GC). 

Lipid extraction
Lipids were extracted from liver tissue using the approach established by Hara and Radin (1978), in which aliquots containing lipids were esterified in 2% methanolic sulfuric acid (Christie, 1989). GC was used to assess fatty acid composition.

Determination of fatty acid composition
To identify and quantify fatty acid methyl esters, 1 mL of n-hexane was added to each sample. Samples were then vortexed and placed in vials. A Shimadzu GC-2010 plus chromatograph FID, an AOC-20i auto-injector, and an autosampler were used for GC analysis. Fatty acids were separated using Permabond (Machhery–Nagel, Germany) silica-coated capillary columns 60 m×0.25 mm I.D., 0.20 μm (Supelco, Sigma, USA). After the column temperature reached 120°C, it was adjusted to 220°C at a rate of 5°C/min, then held constant at 220°C for 15 minutes. Temperature settings for the injector and FID were 240°C and 280°C, respectively. Nitrogen was used as the gas phase, with a column flow rate of 1.2 mL/min, 43 cm/s. Comparisons were made possible by identifying methyl esters and analyzing standard external mixtures under the same conditions.

Determination of lipophilic vitamins
Tissue samples were homogenized by adding 5 mL of isopropyl alcohol/hexane (2:3: v/v) per gram. Samples were then vortexed and centrifuged for 10 minutes at 6000 rpm at 4°C. Supernatants were collected and evaporated with nitrogen before adding 1 mL of an equal volume of acetonitrile/methanol mixture to each container. A SUPELCOSILTM LC-18-DB HPLC column (250×4.6 mm, 5 μm) was employed to examine lipophilic vitamins. A mixture of 75% acetonitrile and 25% methanol was used as the mobile phase. The mobile-phase flow rate was maintained at 1 mL/min. The column’s temperature was maintained at 30°C. Detection wavelengths were 320 nm for retinol (vitamin A) and 215 nm for vitamins K1, tocopherol (Toc), D2, and D3. Analysis results were presented as g/g (Katsanidis & Addis, 1999).

Cholesterol analysis
Isopropanol/ethanol/acetonitrile (30:70, v/v) was used to extract liver samples. The extracts were vortexed and centrifuged at 6000 g and 4°C for ten minutes. Aliquoted supernatants were deposited in vials for HPLC analysis (Shimadzu). The mobile phase was a mixture of isopropyl alcohol and acetonitrile (30:70 v/v). The flow rate was set at 1 mL/min (Bragagnolo & Rodriguez, 2003). HPLC analyses were conducted using a SUPELCOSILTM LC-18 250×4.6 mm, 5 μm column. Detection was made by UV absorption at 202 nm, and column oven temperature was set at 30°C (Katsanidis & Addis, 1999) tocotrienols, and cholesterol in muscle tissue were developed. Method A involves basic saponification of the sample, but causes losses of the γ- and δ-homologs of vitamin E. Method B does not involve saponification, thereby protecting the more sensitive homologs. Both permit rapid analysis of multiple samples and neither requires specialized equipment. These methods may provide techniques useful in simultaneous assessment of oxidative stress status (OSS. Class VP software was used to quantify external standardization. 

HPLC and malondialdehyde analysis
Tissues were homogenized in a homogenizer by adding 1 mL of pure water and 1 mL of 10% perchloric acid to 0.5 g of liver tissue. Homogenized samples were placed in the centrifuge tubes (ISOLAB Garmany) and centrifuged at 10000 rpm for 5 min. Supernatants were removed and placed in vials for HPLC analysis. The malondialdehyde (MDA) mobile phase solution consisted of 0.05 g of dihydrogen phosphate, 110 mL of methanol, and 500 mL of purified water. Phosphoric acid was added to the MDA mobile phase solution to adjust pH to 3.50. Amounts determined in these analyses were reported in nmol/g.
The Karatepe method was modified for malondialdehyde analysis. A C18-ODS-3.5 μm, 4.6×250 mm column, and UV-Vis SPD-10 AVP detector for Shimadzu HPLC were used to measure MDA (Sedlak & Lindsay, 1968).

Glutathione analysis
Glutathione (GSH) content was determined using a UV spectrophotometer with a modified Sedlak and Lindsay method against pure water at a wavelength of 412 nm (Sedlak & Lindsay, 1968).

Statistical analyses
SPSS software, version 16 was used for statistical analysis. Experimental results are reported as Mean±SD and compared with control group results using analysis of variance (ANOVA) and the LSD test.

Results

Liver fatty acid compositions 
All experimental groups were compared with the control group (Table 1). Oleic acid (18:1, n–9c) and eicosatrienoic acid (20:3, n-3) levels increased in the F, F+HPsP, and double-dose groups (P<0.0001, P<0.01, P<0.0001, P<0.05, respectively). Behenic acid (C 22:0) levels increased in the F and F+HPsP groups (P<0.0001 and P<0.01, respectively). Alpha-linolenic acid (18:3, n-3) and arachidonic acid (20:4, n-6) levels decreased in the F and F+HPsP groups (P<0.0001 and P<0.01, respectively). Alpha-linolenic acid (18:3, n-3) levels also decreased in the double-dose group (P<0.0001). Gamma linoleic acid (18:3, n-6) and docosahexaenoic acid (22:6, n-3) levels decreased in the F+HPsP group (P<0.05, P<0.001, respectively). Eicosatrienoic acid (20:3, n-3) levels were significantly increased in both groups (P<0.0001 and P<0.01, respectively). Docosadienoic acid (22:2, n-6) levels were significantly decreased in the double-dose group (P<0.05) but did not show any significant change in the other groups. No significant changes were observed in levels of eicosatrienoic acid (20:3 n-6) and lignoceric acid (24:0) in any groups (P>0.05). Arachidonic acid (20:4 n6) levels decreased in the F and F+HPsP groups (P<0.001, P<0.01, respectively). Eicosapentaenoic acid (20:5, n-3) levels increased in the double-dose group and decreased in the other groups (P<0.0001, P<0.0001, P<0,001).

Liver cholesterol, lipophilic vitamins, MDA, and GSH levels
Liver tissue cholesterol levels increased remarkably in the F, HPsP, F+HPsP, and double-dose groups compared to the controls (P<0.05, P<0.01, P<0.001, and P<0.0001, respectively). Increased retinol levels were observed in the HPsP group (P<0.05). Vitamin K-1 levels increased in the F, HPsP, F+HPsP, and double-dose groups (P<0.05, P<0.0001, P<0.001, and P<0.0001, respectively), and vitamin K2 levels decreased in the HPsP, F, F+HPsP, and double-dose groups (P<0.05, P<0.0001). Vitamin D3 levels decreased in the F, F+HPsP, and double-dose groups (P<0.01, P<0.0001). Levels of α-Toc levels markedly reduced in the double dose group (P<0.05). MDA levels were considerably higher in the F and HPsP groups (P<0.0001) and did not change in the other groups. GSH levels decreased markedly in the F, HPsP, and F+HPsP groups (P<0.0001 and P<0.01, respectively) and increased appreciably in the double-dose group (P<0.0001) (Table 2).

Discussion
Helichrysum species produce flavonoids, acetophenones, phloroglucinols, pyrones, triterpenoids, and sesquiterpenes as secondary metabolites. Flavonoids are critically important phenolics that act as free radical scavengers in higher plants. Some plant species that are exceptionally rich in flavonoids are used to treat elevated free radical-mediated diseases, including NAFLD with excessive liver fat levels (Acet et al., 2020; Aguilera et al., 2016; Sun & Qu, 2019).
Lipid-reducing treatments are recommended for NAFLD patients, with 10% weight loss recommended in the first stage. Due to lifestyle changes, a complete treatment protocol has not yet been developed. Several scientific studies have been conducted on antioxidant and phytotherapeutic effects (Guvenc et al., 2017; Choi et al., 2019). Although the initial stages of this disease are not typically severe, death may occur in later stages due to fluid retention, increased inflammation, and cirrhosis. Many studies have investigated NAFLD caused by excessive fructose consumption (Choi et al., 2019).
In this study, with the application of HPsP, the 16:1 to 16:0 ratio, as a sign of Δ9 stearoyl-coenzyme A desaturase (SCD) synthesis, surprisingly decreased in the fructose group and increased in F+HPsP and double dose groups. The insulin resistance promoted by palmitic acid (16:0) is reversed by palmitoleic acid (16:1, n-9) (Puri et al., 2007). Stearic acid (18:0) is turned over to oleic acid (18:1 n-9) by Δ9 desaturase. Δ9 desaturase activity is raised in HPsP and double dose groups. The decrease in palmitoleic acid (16:1, n9) and the increase in its advanced product, vaccenic acid (18:1, n7), are signs of enhanced Δ9 SCD synthesis.

Reduced stearic acid (18:0) and palmitoleic acid (16:1, n-9) levels in fructose groups may be in response to fatty acid accumulation as a sign of increased lipogenesis in F and F+HPsP groups compared with the control. A rise in the monounsaturated fatty acid/saturated fatty acid ratio is related to increased lipogenesis (Puri et al., 2007).
Linoleic (18:2, n-6c) and linolenic (18:3, n-3) acids are the first substrates of n6 and n3 polyunsaturated fatty acid (PUFA) metabolism, and are essential PUFAs. Linoleic acid levels (cis-18:2, n-6) decreased in the F, F+HPsP, and double-dose groups (P<0.0001). HPsP affected gamma-linolenic (18:3, n-6) and dihomo-γ-linolenic (20:3, n-6) acids, the advanced metabolites of linoleic acid (18:2, n-6) which requires Δ6 desaturase (Δ6DS) for synthesis. Δ6DS activity is affected by HPsP treatments to varying degrees. It causes an increase in the γ-linolenic acid to linoleic acid (18:3, n-6/18:2, n-6) ratio in fructose groups (P<0.05) (Guvenc et al., 2017; Ghazali et al., 2020).
Dihomo-γ-linolenic acid (20:3, n-6) levels also support these changes. Levels of their advanced product, the essential fatty acid arachidonic acid (20:4, n-6), were elevated in the F+HPsP and double dose groups. Phospholipase A2 releases arachidonic acid (20:4, n-6) from cell membranes, and phospholipase C releases arachidonic acid from phosphatidylinositol bisphosphate. The released arachidonic acid (20:4, n-6) is converted into pro-inflammatory prostaglandins, thromboxanes, and leukotrienes. The increased synthesis of leukotrienes and inflammatory prostaglandins can be explained by the elevated consumption of arachidonic acid (20:4, n-6) in the fructose groups. The increase in arachidonic acid (20:4, n-6) levels in the F+HPsP group was surprising (Guvenc et al., 2017; Ghazali et al., 2020).
Surprisingly, n-3 fatty acid changes were also detected. Linolenic acid (18:3, n-3), eicosapentaenoic acid (20:5, n-3), and docosahexaenoic acid (22:6, n-3) decreased in the fructose group. HPsP increased eicosapentaenoic (20:5n-3) acid levels but did not affect linolenic (18:3, n-3) and docosahexaenoic (22:6, n-3) acid levels. Docosahexaenoic acid (22:6, n-3) and eicosapentaenoic acid (20:5, n-3) have anti-inflammatory, antiproliferative, immunomodulatory, and metabolic effects (Guvenc et al., 2017; Ghazali et al., 2020).
The diet’s increased omega-6 to omega-3 fatty acids ratio exacerbates NAFLD development (Allard et al., 2008). The n-6/n-3 ratio of F, F+HPsP, and double-dose groups also decreased. Allard et al. (2008) reported reduced levels of hepatic n-6 and n-3 PUFAs, as also noted by Allard et al. (2008). HPsPs increased cholesterol levels in a dose-dependent manner. However, the mechanism remains unclear.
The anti-inflammatory and antifibrotic properties of Vitamin D and its role in cell regulation and differentiation likely function via metabolic processes. Vitamin D deficiency is associated with insulin resistance and metabolic syndrome (Kitson & Roberts, 2012; Rhee et al., 2013; Kaywanloo et al., 2022). Vitamin D levels in the groups receiving F decreased, similar to previous studies compared to those in the control group. This decrease in vitamin D levels may be due to a decrease in vitamin D-binding protein levels. HPsPs do not affect liver vitamin D levels (Rhee et al., 2013; Jablonski et al., 2013).
As an electron donor, GSH is the most important cellular antioxidant, which scavenges free radicals in its reduced form. Decreased GSH levels correlate strongly with increased free radical production, increasing oxidative stress. Decreased GSH levels, antioxidant activity, and accumulation of hepatic inflammation are essential sources of ROS overproduction in NAFLD (Liu et al., 2015). Increased oxidative stress due to low GSH levels may cause oxidative modification of lipoproteins. This modification can accumulate cholesterol and triglycerides, causing insulin resistance (Jain et al., 2014).
The antioxidant defense system is weakened by the overproduction of free radicals that deplete antioxidant levels via lipid peroxidation, making the liver susceptible to oxidative damage. MDA levels, which can indicate lipid peroxidation, were considerably higher in the F and HPsP groups than in the control groups, as expected from the results of other studies, and the application of HPsP improved MDA and GSH levels. In particular, the double dose significantly increased GSH levels. Toc levels indicate free radicals (Kitson & Roberts, 2012; Serviddio et al., 2013).
Vitamin E is an effective chain-breaking antioxidant that scavenges peroxyl, superoxide, and hydroxyl radicals. It also protects cells against lipid peroxidation. Vitamin E consists of tocopherols (α, β, γ, δ) and tocotrienols (α, β, γ, δ). α-Toc is the most abundant tocopherol and is important in inhibiting lipid oxidation. In cell membranes, low-density lipoprotein and polyunsaturated lipids are protected from oxidation by α-Toc (Li et al., 2016).
Decreased antioxidant defense levels have been generally observed in liver disease. Decreased α-Toc levels in liver diseases have been observed, consistent with our results, and HPsP has demonstrated no effect on α-Toc levels (Di Sario et al., 2007). Vitamin E supplementation slows the progression of NAFLD to NASH (Elizabeth et al., 2010). Fructose application decreased Toc (important in clearing free radicals during metabolism) levels in all groups. Surprisingly, HPsP did not show the expected effect.
Vitamin K is a fat-soluble vitamin formed from phylloquinone (K1) and menaquinone (K2) synthesized by intestinal bacteria. Menaquinones are of bacterial origin and are produced by microbiota (exp., the Firmicutes). The distal intestine synthesizes endogenous vitamin K2 (Knapen et al., 2018; Wagatsuma et al., 2019). Vitamin K2 is mainly found in fermented milk products and is produced by lactic acid bacteria in the gut. Phylloquinone (K1) is of plant origin and binds to the membranes of plant chloroplasts. In animals, vitamin K3 (menadione) is metabolized to vitamin K2. Vitamin K3 is a synthetic vitamin added to animal feed as a source of vitamin K (Wagatsuma et al., 2019). Leafy green vegetables, including soybeans, cottonseed, canola, and olives, are good sources of vitamin K and may be consumed by overweight and obese individuals (Booth & Suttie, 1998; Kimmons et al., 2006). Vitamin K positively influences glucose regulation. Decreased insulin resistance is associated with elevated plasma levels of vitamin K1 (Varsamis et al., 2021).
Vitamin K deficiency is associated with increased body fat, and triglyceride-rich lipoproteins elevate circulating vitamin K1 levels (Shea et al., 2010). Low vitamin K levels decrease urinary gamma-carboxyglutamic acid excretion (Ferland et al., 1993). Owing to its ability to increase insulin production and sensitivity, diets containing high amounts of vitamin K are associated with a decreased incidence of type 2 diabetes mellitus. Another study has suggested that vitamin K2 intake may benefit metabolism (Booth, 2012). NAFLD is often associated with metabolic syndrome. The leading phyla in the gastrointestinal microbiota are Firmicutes, Bacteroidetes, and Proteobacteria. The elevated triglyceride levels and central obesity significantly reduced diversity among these phyla. Steatosis and obesity have been linked with the Firmicutes/Bacteroidetes ratio. NAFLD increases Firmicutes that may cause hepatic steatosis by regulating fatty acid inflow and lipogenesis, potentially exacerbating hepatic steatosis (Raman et al., 2013; Chen et al., 2019).
In all groups receiving HPsP, K1 vitamin levels were elevated compared to the control group. Vitamin K2 levels decreased in the F and HSP groups. Currently, the effect of microbial diversity on intestinal bacteria has remained unclear. HPsP may also increase metabolic activity. The mechanism underlying the decrease in vitamin K2 levels in the F group is unknown but may involve the firmicute/bacteria ratio, which decreases with obesity or NAFLD.
The chemical composition and other properties of HPsP in Turkey have been investigated by several researchers (Demir et al., 2009; Taşkın et al., 2020). HPsH produces compounds such as helichrysums A and B, apigenin, naringenin, isoastragalin, isosalopurposide, isoquercitrin, dicaffeoylquinic acid, and luteolin. Apigenin protects against nonalcoholic fatty liver disease by decreasing lipid accumulation and ameliorating insulin resistance (Jung et al., 2016). This study is the first to elucidate biochemical parameters for fructose-induced HPsP, including the liver’s lipophilic vitamins and fatty acid composition. The study also demonstrated that HPsP application increases liver total cholesterol. Taken together, our results provide experimental confirmation of the health value of HPsP application to fructose-induced rats. The mechanisms by which HPsP application increases liver cholesterol are unclear and require further research.

Ethical Considerations

Compliance with ethical guidelines
This study was approved by the Animal Ethics Committee of Fırat University (2014/122) and performed in accordance with the rules of the European Economic Community (EEC, 1986). 

Funding
This research is extracted from the master’s thesis of Ibrahim Dogan, approved by Department of Biology, Faculty of Science, Adıyaman University, and was financially supported by Department of Scientific Research Project Management of Adıyaman University (ADYUBAP) (Project No.: 2014-0005).

Authors' contributions
Conceptualization, supervision, investigation and writing-original draft: Mehmet Guvenc, and Ibrahim Dogan; Writing–review & editing: Mehmet Guvenc; Data collection: Ibrahim Dogan.

Conflict of interest
The authors declared no conflicts of interest.

Acknowledgments
The authors thank the Department of Scientific Research Project Management of Adıyaman University (ADYUBAP) for providing financial assistance. The authors also appreciate Kazim Sahin for his advice

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